PURGING OF BONE MARROWS CONTAMINATED WITH MYELOID LEUKEMIC CELLS BY INTERLEUKIN-2 AND LYMPHOKINE-ACTIVATED KILLER CELLS

The capability of recombinant human interleukin-2 ( rhIL-2) and lymphokine-activated killer (LAK) cells in the purging of normal human bone marrows contaminated with human myeloid leukemic cell lines was evaluated. Mixtures of normal human bone marrow mononuclear cells ( BMC) and K562 cells or HL-60 cells (at the BMCK562 ratio of 200:1, 100:1 or 20:1) were incubated with IL-2 with or without LAK cells at the BMC:LAK ratio of 1:1 for one or three days. The nubmers of residual K562 cells, BFU-E and CFU-GM were examined by clonogenic assays. In 200:1 mixture groups without LAK cells, the number of K562 colonies reduced by 50% with no loss of BFU-E and CFU-GM in one-day cultures, and no K562 colonies formed in three-day cultures with about 20% loss of BFU-E and CFU-GM. If the BMC.K562 ratios were 100:1 or 20:1 in the mktures, the leukemic cells could not be eliminated. When the mixtures were incubated with IL-2 and LAK cells, no leukemic cell colonies were detected in the 20:1 group following one-day

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

THE EFFECT OF PHENYLACETATE ON THE EXPANSION AND CYTOTOXIC ACTIVITY OF ADHERENT LAK CELLS FROM PATIENTS WITH HEPATOCELLULAR CARCINOMA

Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

AUGMENTATION OF IMMUNE FUNCTIONS AND AUTOLOGOUS TUMOR-KILLING ACTIVITY BY KAPPA-SELENOCARRAGEENAN IN MICE BEARING SARCOMA 180

Objective: To study the enhancement of the immune functions and autologous tumor killing (ATK) activity by kappa selenocarrageenan (KSC) in mice bearing sarcoma 180. Methods: To measure the effects of KSC and/or Cyclophosphamide (Cy) on natural killer (NK) activity, lymphokine activated killer (LAK) activity, the produc tion of interleukin 2 (IL 2), ATK activity and the growth of sarcoma 180 (S 180 ). Results: KSC promoted NK activity, LAK activity and ATK activity in vivo , increased IL 2 production at 40 mg/kg/d×9d. It also enhanced the antitumor action of Cy (20 mg/kg/d×9d) and offset the inhibition of Cy on immunocopetent cells. The ATK activity in splenocytes of S 180 bearing mice could be induced and increased by recombinant interleukin 2 (rIL 2) in vitro . Conclusion: KSC has an up regulating effect on the immune functions and ATK activity in tumor bearing mice. It can be used as a biological response modifier (BRM) in cancer biotherapy.

THE ULTRASTRUCTURAL OBSERVATION OF TARGET CELLS ATTACKED BY HUMAN LAK

In order to explore the killing mechanism of LAK, we observed the morphological change of K562 and Raji attacked by human LAK with transmission electron microscope, The result showed that 1 hour after coculture of LAK and target cell, target cell was significantly damaged.Part of target cells died via necrosis, and part via apoptosis. Our findings show that human LAK can kill target cells via necrosis and apoptosis simultaneously

RESEARCHES ON THE ANTI-AUTOLOGOUS TUMOR EFFECTS OF HUMAN CANCER-KILLING CELLS IN NUDE MICE

An investigation of human splenic LAK cells killing autologous tumor in vivo has been performed in this study. Briefly, an ovary embryo carcinoma (OEC) removed surgically from patient was transplanted Into nude mice (s. c. ) and cultured in vitro, that would be used as targets. Lymphokine-activated killer (LAK) cells were generated from splenic lymphocytes of the OEC patient, who died two and half months after operation, by co- culture with recombinant human interleukin- 2 ( rIL- 2 ) in vitro. The results from Winn' s test in nude mice suggested that these LAK cells could effectively inhibit the tumorlgenicity of autologous tumor m vitro.

TREATMENT OF PATIENTS WITH MALIGNANT PLEURAL EFFUSIONS DUE TO ADVANCED LUNG CANCER BY TRANSFER OF AUTOLOGOUS LAK CELLS COMBINED WITH rIL-2 OR rIL-2 ALONE

Experimental study both in vitro and in vivotogether with clinical trials showed that LAKcells have antitumor and antimetastatic effects(1-5)and that these effects are closely related tothe number of LAK cells transferred and the ad-ministration of rIL-2(1,6-8).Usually,autologousPBL’s are used as the source of LAK precursorsin the adoptive immunotherapy of cancer patients.But this not only puts an added burden on thecancer patient,it can cause serious side effectsas well(9).Although TIL’s may provide a solu-tion to this problem(10,11),their isolation fromsolid tumors is complex and consumes many rea-gents.We have reported that the isolation oflymphocytes from malignant ascites or from ma-lignant pleural effusions is not only simple

Modulating effect of mitomycin or cisplatin on lymphokine-activated killer cell proliferation and antitumor activity to bladder cancer cell lines in vitro

Ｍｏｄｕｌａｔｉｎｇｅｆｅｃｔｏｆｍｉｔｏｍｙｃｉｎｏｒｃｉｓｐｌａｔｉｎｏｎｌｙｍｐｈｏｋｉｎｅａｃｔｉｖａｔｅｄｋｉｌｅｒｃｅｌｐｒｏｌｉｆｅｒａｔｉｏｎａｎｄａｎｔｉｔｕｍｏｒａｃｔｉｖｉｔｙｔｏｂｌａｄｄｅｒｃａｎｃｅｒｃｅｌｌｉｎｅｓｉｎ...

Tretinoin or retinol enhancement of lymphokine-activated killer cell proliferation and cytotoxicity against human bladder cancer cells in vitro

目的：研究维Ａ酸（Ｔｒｅ）或维生素Ａ（Ｒｅｔ）对膀胱肿瘤患者淋巴因子激活的杀伤细胞（ＬＡＫ细胞）的增殖和对膀胱肿瘤细胞的细胞毒作用．方法：分别用细胞计数和ＭＴＴ法测定ＬＡＫ细胞的增殖和细胞毒作用．结果：Ｔｒｅ或Ｒｅｔ１０－１００ｎｍｏｌ·Ｌ－１加强由白细胞介素２（ＩＬ２）诱导的ＬＡＫ细胞的增殖和对膀胱肿瘤细胞的杀伤作用．结论：Ｔｒｅ或Ｒｅｔ增强膀胱肿瘤患者ＬＡＫ细胞增殖及对膀胱肿瘤的细胞毒作用．

参麦注射液对晚期癌症患者sIL－2R、LAK及NK细胞活性影响

本研究测定了６０例晚期癌症患者用参麦注射液治疗前后血清可溶性白细胞介素－２受体（ｓＩＬ－２Ｒ）、淋巴因子活化的杀伤细胞（ＬＡＫ）和天然杀伤细胞（ＮＫ）的活性，并以４０例正常人为对照。结果显示：治疗前癌症患者ｓＩＬ－２Ｒ显著升高（Ｐ＜０．０５），ＬＡＫ细胞与ＮＫ细胞活性显著减低（Ｐ＜０．０５）。而上述变化在胃癌、肠癌与肺癌之间无显著差异（Ｐ＞０．０５）。治疗后３种癌症患者的ｓＩＬ－２Ｒ均有显著下降（Ｐ＜０．０５），ＬＡＫ与ＮＫ细胞活性均有显著增强（Ｐ＜０．０５）。而ｓＩＬ－２Ｒ与ＬＫＡ及ＮＫ细胞活性之间无相关性。提示晚期癌症患者免疫功能低下，参麦注射液对癌症患者的免疫功能有广泛的调节作用。

人参皂苷Rg1、Rh1对树突状细胞刺激T细胞增殖及LPAK抗肿瘤活性的影响

目的 :观察人参皂苷Rg1,Rh1对树突状细胞 (DC)刺激T细胞增殖及其对IL 2与PHA激活的杀伤细胞 (DC LPAK)对人乳头瘤细胞及L92 9细胞杀伤作用的影响。方法 :分离培养鉴定DC ,用MTT法检测Rg1及Rh1对DC刺激T淋巴细胞增殖作用的影响。用中性红摄入比色法观察DC LPAK的杀伤活性。结果 :T :DC为 10 :1及 2 5 :1时 ,仅Rg110 0 μg ml能促进T细胞增殖 (P <0 0 5 ) ,而Rh110 0 μg ml则抑制T细胞增殖 (P <0 0 1) ,同剂量的Rg1及Rh1相比有显著性差异 (P <0 0 5～0 0 1) ;T :DC为 10 :1时 ,Rh110 μg ml抑制T细胞增殖 ,而在T :DC为 2 5 :1时则表现为促增殖作用 (P <0 0 5 )。T :DC为 5 0 :1时 ,各加药组对T细胞增殖均无作用。Rg1及Rh1均能促进DC LPAK对乳头瘤细胞的杀伤能力 ;在对L92 9杀伤实验中 ,效靶比为 5 :1时 ,Rg1各剂量均能显著促进DC LPAK的杀伤活性 (P <0 0 1～ 0 0 5 ) ,而Rh1仅中剂量能提高DC LPAK的杀伤活力 (P <0 0 5 )。结论 :Rg1可增强DC刺激T细胞增殖及DC LPAK杀伤肿瘤的细胞能力。Rh1的作用则与其剂量范围及细胞比例密切相关。

猪苓多糖、香菇多糖和分枝杆菌多糖对淋巴因子活化杀伤细胞活性增强作用的研究

本实验对猪苓多糖（ＰＰＳ）、香菇多糖（ＬＥＮ）和分枝杆菌多糖（ＭＰＳ）在体外对淋巴因子活化杀伤（ＬＡＫ）细胞活性的影响进行了研究，将三种不同浓度的多糖与不同浓度的重组白细胞介素２（ｒＩＬ－２）一起共同培养健康人外周血单个核细胞（ＰＢＭｃ），９６小时后以氚－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入法细胞毒试验检测ＬＡＫ细胞对不同靶细胞（包括ＮＫ敏感和ＮＫ抵抗）的体外杀伤活性。结果发现：三种多糖在一定浓度范围内与ｒＩＬ－２合用能增加ＬＡＫ细胞活性４２％～５６．９％，降低ｒＩＬ－２用量５０％（Ｐ＜０．０５～０．０１）。结果提示：三种多糖均可作为细胞活性上向调节剂（ＬＵＲＡ）用于ＬＡＫ细胞疗法。

自体CIKs治疗对乙肝病毒的抑制作用及机制分析

目的探讨细胞因子诱导的杀伤细胞(CIKs)治疗对乙肝病毒(HBV)的抑制作用及其机制。方法选取2009年4月-2010年12月于解放军302医院住院,且未经抗病毒药物及其他免疫治疗的慢性乙肝患者16例,采集外周血单个核细胞(PBMC)经细胞因子鸡尾酒诱导培养成CIK细胞后,经静脉回输患者体内。观察CIK细胞治疗后24周内患者体内HBV DNA水平、CD3+CD56+细胞频率的变化,以及髓样树突状细胞(mDCs)和浆样树突状细胞(pDCs)频率的改变。结果自体CIK回输后,9例患者产生病毒学应答,其体内HBV DNA水平分别于第41、2及24周(分别为5.70、5.09和4.08log10拷贝/ml)出现明显降低(P<0.05或P<0.01);7例患者表现为病毒学无应答,其体内HBV DNA水平均与在基线时(6.39log10拷贝/ml)无明显差异(P>0.05)。经14d的诱导培养后,病毒学应答者CIK细胞的主要效应细胞CD3+CD56+细胞比例为17.21%,明显高于无应答者(8.97%,P<0.05)。经CIKs治疗后,病毒学应答者pDCs频率由治疗前的0.27%上升至治疗后4周的0.39%(P<0.05)和治疗后12周的0.34%(P<0.05),而病毒学无应答者无明显改变。CIK细胞回输后无明显不良反应。结论 CIK细胞治疗慢性乙肝安全性好,能抑制HBV复制,其机制部分是通过提高pDCs频率实现的。

氧化苦参碱对LAK细胞活性的影响

ＬＡＫ细胞具有很强的广谱杀瘤作用，而氧化苦参碱具有较强的免疫抑制作用。本文研究了氧化苦参碱对ＬＡＫ细胞活力的影响，结果表明：氧化苦参碱可抑制ＩＬ－２对小鼠脾细胞的促增殖作用。并且对ＩＬ－２活化ＬＡＫ细胞杀伤Ｐ８１５的能力也有抑制作用。当ＩＬ－２（５００ｕ／ｍｌ）与２００μｇ／ｍｌ的氧化苦参碱共同孵育４ｄ后，可使ＬＡＫ细胞杀瘤能力（在效靶比为１００：１时）的８２．５％被抑制。同时氧化苦参碱本身对Ｐ８１５的增殖无任何效应。

淋巴细胞Fas和配体(FasL)表达变化及其与抗肿瘤活性的关系

为探讨淋巴细胞表面Ｆａｓ及其配体（ＦａｓＬ）在ＩＬ－２和ＩＬ－１２作用下表达的变化，以及Ｆａｓ和ＦａｓＬ与淋巴细胞抗肿瘤活性的关系，作者用免疫荧光标记和流式细胞仪分析了ＩＬ－２和ＩＬ－１２激活的杀伤细胞（ＩＬ－２ＡＫ和ＩＬ－１２ＡＫ）Ｆａｓ和ＦａｓＬ的表达；分别用乳酸脱氢酶（ＬＤＨ）释放法和ａｎｎｅｘｉｎ－Ｖ－ＦＩＴＣ标记检测ＩＬ－２ＡＫ和ＩＬ－１２ＡＫ杀伤的肿瘤细胞的坏死和凋亡。结果表明：①正常人外周血淋巴细胞（ＰＢＬ）（２０．４０±５．６９）％表达Ｆａｓ，但无ＦａｓＬ表达；３株消化道肿瘤细胞系恒定表达Ｆａｓ，始终不表达ＦａｓＬ；②ＩＬ－２与ＩＬ－１２可诱导ＬＡＫ细胞表达ＦａｓＬ（Ｐ＜０．０１），并促使Ｆａｓ表达显著增加（Ｐ＜０．０１）；③ＬＡＫ细胞致肿瘤细胞死亡中ＦａｓＬ表达与ａｎｎｅｘｉｎ－Ｖ标记阳性率和肿瘤细胞表面Ｆａｓ表达正相关。由此推论，Ｆａｓ－ＦａｓＬ途径是ＩＬ－２与ＩＬ－１２诱导活化的ＬＡＫ细胞杀伤肿瘤的途径之一，淋巴细胞致肿瘤凋亡分别与淋巴细胞ＦａｓＬ表达和肿瘤细胞Ｆａｓ表达一致。

石斛多糖增强脐带血和肿瘤病人外周血LAK细胞体外杀伤作用的研究

目的：探讨石斛多糖（ dendrobium candidum polysaccharide,DCP）与 rIL- 2联合应用对脐带血 LAK细胞（ CB-LAK）和肿瘤病人外周血 LAK细胞（ PB-LAK）体外杀伤肿瘤细胞作用的影响。方法：应用 3H-TdR释放法测定 CB-LAK和 PB-LAK细胞的杀伤活性。结果：① rIL-2(500 u/ml)能明显提高 CB-LAK对 Raji细胞的杀伤活性 ,0 h的 CB-LAK活性为 13.63％ ,rIL-2(500 u/ml)诱导 48、 72、 96、 120 h后 ,CB-LAK活性分别提高至 22.28％、 26.23％、 28.55％和 25.76％ (P0.05）；③ DCP（ 200 mg/L）与 rIL-2（ 500 u/ml）联合培养 96 h，显著增强 PB-LAK杀伤活性。 10例恶性肿瘤病人外周血（ DCP＋ rIL-2）组与单纯 rIL-2组诱导的 PB-LAK平均活性分别为 34.78％和 23.24％ 。

肺瘤平膏及其拆方对DC刺激LPAK抗肿瘤活性的影响

目的比较不同中药含药血清对树突状细胞(dendritic cell,DC)刺激LPAK抗肿瘤活性,为指导临床用药提供初步的实验依据。方法从健康人外周血中分离获得PBMC,采用多种细胞因子诱导,获取DC及LPAK,采用中性红摄入比色法检测肺瘤平膏及其拆方含药血清干预DC-LPAK细胞杀伤肿瘤细胞活性的不同。结果 LPAK∶Tumor(L∶T)为10∶1或5∶1组时,各中药组DC诱导的LPAK细胞,杀伤活性明显高于对照组(P<0.05)。组内杀伤活性比较,即L∶T(10∶1)与L∶T(5∶1)比较,解毒组、空白DC对照组、T+LPAK组,P<0.05,余各组比较,P>0.05;肺瘤平膏组、益气组、活血组在L∶T为5∶1与10∶1时,诱导LPAK的杀伤活性基本相同。结论肺瘤平膏、活血药、益气药可不同程度增强DC-LPAK杀伤肿瘤细胞的作用,而解毒药则对其有抑制作用。

一种高活力分离肿瘤浸润淋巴细胞方法的建立

肿瘤浸润淋巴细胞（ＴＩＬ）的增殖力、活力、杀伤力是ＴＩＬ临床治疗的基本问题。由于Ｒｏｓｅｎｂｅｒｇ方法存在着许多不足之处。本实验室对其一些重要步骤进行了改进：①用单一冷胶原酶消化肿瘤组织块；②省去影响ＴＩＬ活力的离心操作；③ＴＩＬ培养４８ｈ内除去掺杂在ＴＩＬ中的肿瘤细胞等。由于除去了影响ＴＩＬ活力的抑制因素，ＴＩＬ的增殖率、生存率、活力、杀伤力明显增加。有８０％肿瘤组织的ＴＩＬ经扩增可超过１×１０￣９细胞数，生长周期曲线反映了冷酶法的增殖率超过Ｒｏｓｅｎｂｅｒｇ方法。本方法的建立，为ＴＩＬ克隆、建株打下了基础，显示了ＴＩＬ临床治疗潜能的提高。

巨细胞病毒感染时免疫功能的变化及其意义

目的 探讨巨细胞病毒感染时免疫变化及其意义。方法 以 2 0名CMV IgM(Cytomegalovirus immunoglobulinM)阳性的病儿为观察对象 ,另设对照组 30例。ELISA法检测柯萨奇B组病毒抗原 (CVB Ag)、IgM抗体 (CVB IgM)、巨细胞病毒IgM抗体 (CMV IgM)、EB病毒IgM抗体 (EBV IgM) ,单克隆抗体标记间接ABC免疫法检测外周血T细胞亚群。结果 CMV感染组的LAK细胞、NK细胞活性均明显降低 (P <0 .0 1)。合并其它感染原的CMV混合感染组CD3含量减少 ,CD8含量增加 (P <0 .0 5 ) ,CD4 CD8数值明显降低 (P <0 .0 1)。病毒混合感染并合并支原体感染组CD3含量减少 (P <0 .0 5 )、CD4 CD8数值、IgA含量明显降低 (P <0 .0 1)。结论 巨细胞病毒感染影响了T细胞亚群的平衡 。

抗肿瘤多价转移因子对肿瘤患者NK及LAK活性的影响

目的 制备并观察抗肿瘤多价转移因子 (M TF )对肿瘤患者NK及LAK活性的影响。方法 分别用肝癌细胞悬液、胃癌细胞悬液免疫山羊 ,取其淋巴组织经匀浆透析法制备肝癌特异性转移因子 (HCC STF)和胃癌特异性转移因子 (GC STF) ,用肝癌、胃癌及大肠癌混合细胞悬液免疫山羊 ,制备M TF。比较其理化性质及对肿瘤患者NK及LAK活性的影响。结果 三种转移因子理化性质十分相似 ,肽类、核苷酸类物质及氨基酸含量相近。HCC STF能明显增加肝癌患者NK、LAK活性及LAK细胞对肝癌 772 1细胞的特异细胞毒作用 ,GC STF能明显增加胃癌和大肠癌患者NK、LAK活性及LAK细胞对胃癌 790 1细胞的特异细胞毒作用 ,M TF能显著增加肝癌、胃癌及大肠癌的NK、LAK活性及LAK细胞对 772 1和 790 1细胞的细胞毒作用 ,其增加效果与HCC STF及GC STF比无明显差异。结论 M TF具有免疫调节性强、抗肿瘤谱广的特性 。